Many animal and plant pathogenic bacteria utilize a similar secretion system, termed type Ill or "contact dependent," to deliver a battery of bacterial effector proteins into host cells. Salmonella typhimurium uses such a secretion system to inject proteins that manipulate host cellular functions to induce the uptake of the bacterium into the normally non-phagocytic cells of the intestinal epithelium. This process relies on less than ten translocated effectors proteins, which collaborate to induce dramatic membrane ruffling, leading to bacterial internalization by macropinocytosis. The long-term goal of this work is to use structural biology as a foundation for a molecular understanding of the invasion process of this pathogen, and to exploit this information in the identification of potential targets for drug screening. The specific aims of this proposal are (1) to determine structures of S. typhimurium invasion-associated translocated effectors, (2) to determine the co-crystal structures of these factors with their host cell targets, and, finally, (3) to use structure-based mutagenesis to examine the interacting surfaces of these factors in the context of bacterial host cell invasion and cytoskeletal manipulation. This work will thus involve a multidisciplinary approach combining macromolecular X-ray crystallography, biochemical assays, and microbial cell biology. Bacterial infection is and has been a significant cause of death and human suffering. Ominously, our weapons for combating bacterial pathogens are now failing as ever-increasing numbers of microorganisms have developed resistance to greater numbers of our drugs. Furthermore, the increased threat of the use of microbial agents as instruments of war or terrorism has become a very real concern. Therefore, a final aim of these studies will be to use the structural information to aid in selecting targets for the screening of inhibitory compounds that will impair the virulence mechanisms of this pathogen, and to serve as a paradigm for developing similar strategies against other infectious bacterial organisms.